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Published as doi: 10.1096/fj.06-7659com.
 (The FASEB Journal. 2007;21:1768-1776.)
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From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis

Lars Klinge, Steve Laval, Sharon Keers, Faye Haldane, Volker Straub, Rita Barresi and Kate Bushby1

University of Newcastle, Institute of Human Genetics, International Centre for Life, Newcastle-upon-Tyne, UK

1Correspondence: Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Pkwy, NE1 3BZ Newcastle upon Tyne, England, UK. E-mail: kate.bushby{at}ncl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The dysferlin gene is mutated in limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T-tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+-dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full-length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.—Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., Bushby, K. From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis.


Key Words: membrane repair • muscular dystrophy • T-tubulogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE "MUSCULAR DYSTROPHIES" (MD) are a group of inherited disorders leading to progressive muscle wasting and weakness for which there is currently no curative treatment. Dysferlin is the gene mutated in the autosomal recessive disorders limb-girdle MD type 2B (1) , Miyoshi myopathy (2) , and distal anterior compartment myopathy (3) . Patients present with either proximal or distal weakness in the late teens or early twenties and develop massive elevation of serum creatine kinase activity (4) . Dysferlin is a 230 kDa protein with a C-terminal transmembrane domain. As demonstrated at the ultrastructural level, dysferlin is a sarcolemmal protein (5) , but it is distinct from the dystrophin-glycoprotein complex (DGC) in human muscle (6) . It has recently been shown that a minor proportion of dysferlin is also associated with the T-tubule system in skeletal muscle (7 , 8) , whereas the sarcolemmal labeling is not attributed to peripheral T-tubule profiles or caveolae (5 , 7) . An increasing number of dysferlin interacting proteins have been described including caveolin-3 (9) , affixin (10) , annexins A1 and A2 (11) , calpain 3 (12 , 13) , myogenin (14) , and AHNAK (8) . In numerous types of MD, a secondary displacement of dysferlin to the cytoplasm can be observed (15) , whereas subsarcolemmal accumulation of vesicles is uniquely found in dysferlin deficient skeletal muscle (16) .

Dysferlin is a member of the ferlin family of proteins with at least four human orthologues: dysferlin, myoferlin, otoferlin, and fer1L4 (17) . Otoferlin has been associated with a nonsyndromic form of deafness (MIM 601071) (18) , whereas no disease has been identified for mutations in myoferlin, which is highly expressed in skeletal and cardiac muscle and is required for myoblast maturation (19) . The ferlins are named for their homology to fer-1, a C. elegans protein required for fusion of vesicles with the spermatid cell membrane (20 , 21) . The ferlins are unique in harboring six C2 domains (5 in fer1L4). C2 domains have first been described in protein kinase C (22) , comprise ~130 residues, and are known to bind phospholipids in a Ca2+-dependent manner to act in membrane trafficking, signal transduction, protein-protein interactions, and membrane fusion (23) .

Dysferlinopathies are the first class of diseases identified so far where it has been suggested there is a fault in the process of membrane repair (6) rather than a structural weakness of the sarcolemma (24 , 25) . Wounding of isolated murine dysferlin deficient myofibers by laser irradiation demonstrated a reduced membrane repair activity when compared to isolated wild-type murine myofibers, suggesting a global role of dysferlin in the process of membrane repair (6) . Enrichment of dysferlin at the sites of injury in isolated wild-type murine myofibers is suggestive of a direct role of dysferlin in the process of membrane repair (6) . This role has been proposed to be in vesicle docking and fusion but up to now the precise nature of dysferlin containing vesicle generation is unknown.

To gain further insight into the function of dysferlin in membrane repair, we investigated the subcellular expression pattern of dysferlin in C2C12 myotubes as a cellular model of muscle differentiation and characterized its response to membrane wounding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
C2C12 myoblasts, a model of early myogenesis, were obtained from European Collection of Cell Cultures (Wiltshire, UK) and routinely cultured at subconfluence in DMEM + 15%FCS and penicillin/streptomycin. Differentiation was induced by culturing the cells in DME + 3% horse serum for 4–6 days. Transfections with plasmids described below were carried out using 1–10 µg of plasmid DNA using FuGene-6 lipofection reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. After transfection, myoblasts were allowed to recover for 24 h and attain confluence before differentiation was induced.

Antibodies and reagents
Polyclonal anti-dysferlin antibody (Abcam, Cambridge, UK) and monoclonal anti-Bin1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a dilution of 1:100. A monoclonal anticaveolin-3 antibody (Transduction Laboratories, Oxford, UK) was used at a dilution of 1:1000. Secondary antibodies were anti-rabbit TRITC or FITC and anti-mouse-FITC supplied by DAKO-Cytomation (dilution 1:50). Methyl beta-cyclodextrin, used for the cholesterol depletion to specifically disrupt the T-tubule system, was purchased from Sigma-Aldrich (St. Louis, MO, USA) applied at a concentration of 10 mM for 30 min to the cultured cells before analysis. DNA synthesis was investigated by a 5-bromo-2'-deoxy-uridine (BrdU) assay to determine cell viability. BrdU labeling reagent (Roche, Mannheim, Germany) was added onto the cells at a dilution of 1:1000 in culture medium and incubated for 20 min at 37C. After three 5 min washes with PBS, the cells were fixed for 20 min at –20C in ethanol fixative (50 mM glycine solution to 70 ml abs. EtOH, pH 2.0) and washed three times for 5 min with PBS thereafter. The cells were then covered with anti-BrdU working solution (Roche) and incubated for 30 min at 37C. After three washes with PBS, the cells were incubated with anti-mouse-Ig-fluorescein working solutions (Roche) for 30 min at 37°C and then washed three times with PBS and mounted with Vectashield mounting medium with DAPI. 7-Amino actinomycin D (7-AAD; Pharmingen, Oxford, UK), a fluorescent vital dye that stains DNA and does not cross intact plasma membranes, was used as a marker of cell death and was applied to a monolayer of cells after fixation at 15 µl/ml PBS and incubated for 15 min at 37 C before analysis. Poloxamer 188 (BASF, Ludwigshafen, Germany), a membrane sealing agent, was applied at a concentration of 0.5 mM.

Plasmids
EGFP fusion proteins were constructed in pcDNA4/TO/MycHIS (Invitrogen, Carlsbad, CA, USA) and are detailed diagrammatically in Fig. 2 . GFP-DFL encodes the entire dysferlin sequence with an N-terminal EGFP fusion. The remaining constructs are N-terminal fusions between EGFP and reduced dysferlin sequences. DSC-Xba encodes the cDNA up to the XbaI site (all of C2A and part of C2B). DSC-Xho encodes the cDNA up to the XhoI site (C2A-C, plus part of the dysferlin domain). GFP-Tth encodes the cDNA from the TthI site to the end (dysferlin domain and C2D-F). 3'GFP-2 encodes the cDNA from the StuI site between C2D and C2E to the end. GFP-TM encodes the cDNA from the HindIII site to the end (the transmembrane domain and C-terminus only). GFP-caveolin-3 encodes the entire caveolin-3 coding sequence as a C-terminal EGFP fusion.


Figure 1
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  		Figure 1. Subcellular localization of dysferlin in C2C12 myotubes 6 days after differentiation by confocal microscopy. A) Double immunofluorescence for dysferlin and Bin1 reveals colocalization at the site of contact between a myotube and a myoblast (arrowhead) and also close to sarcolemma on the left-hand side of the cell in structures most likely to be beaded invaginations of the plasma membrane. The image was taken with focus on the site of contact of the 2 cells. Scale bar = 10 µm. B) Wide field (top), and confocal fluorescence imaging (bottom) reveal that dysferlin fails to localize to the plasmalemma (arrowheads), as shown in image in middle (bright field), same cell as on top. Confocal image of a different cell (bottom) is representative of dysferlin expression in C2C12 myotubes. Scale bar = 10 µm. C) Double immunofluorescence demonstrates that endogenous dysferlin colocalizes with Bin1. Scale bar = 5 µm. D) Cholesterol depletion with methyl beta-cyclodextrin disrupts dysferlin and Bin1 localization. In contrast to dysferlin, Bin1 is detectable at the plasma membrane after this treatment. Scale bar = 10 µm.


Figure 2
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  		Figure 2. Targeting of dysferlin to T-tubules requires the full-length protein. A) Domain diagram of dysferlin constructs tested by transient transfection of corresponding GFP fusion proteins and summary of localization results. TM: transmembrane domain. B) GFP fluorescence of C2C12 myotubes expressing constructs depicted in A. Only full-length dysferlin is localized in an intracellular network identified as T-tubules. All images are confocal sections through middle of cells. Scale bar = 10 µm.

Wounding assay
The bead wounding assay was utilized to investigate the consequences of membrane disruption on the subcellular distribution pattern of dysferlin, its deletion mutants, Bin1 and caveolin-3. Bead wounding was applied to create membrane ruptures in the presence of extracellular Ca2+ and absence of extracellular Ca2+ (chelation with EGTA). To study if cells were able to reseal the ruptures sustained during the wounding protocol, DNA synthesis was investigated using a BrdU assay to determine cell viability. Ca2+ dependency of the resealing process after bead wounding was demonstrated by 7-AAD staining and quantification of the immunofluorescence signal. For the wounding assay, 0.1 mg acid-washed glass beads (40–70 µm, Sigma) were washed three times in 20 ml PBS. Thereafter, the beads were resuspended in 1 ml PBS containing lysine-fixable rhodamine dextran (10 kDa, 1 mg/ml in PBS, Molecular Probes, Eugene, OR, USA), a membrane impermeable macromolecule, and dispersed on differentiated C2C12 myotubes growing on slide flasks (Nunc, Wiesbaden, Germany). The slide flasks were gently rocked 20 times to let the beads roll over the cells, rinsed in PBS and either fixed and analyzed immediately or after incubation in medium at 37°C for 3 and 5 min before analysis. For viability assays using BrdU, the cells were incubated for 5 h in medium before analysis. Wounding assays with EGTA (10 mM, Sigma) were performed as described above. Poloxamer 188 was applied at a concentration of 0.5 mM during wounding and also during the following incubation period (2 h). For analysis of survival after bead wounding applying 7-AAD, three different experiments were used. In each experiment, three slides per group were analyzed by taking 20 different random fluorescence images at x10 magnification per slide (rhodamine filter). ImageJ software was applied to measure the area of fluorescence in micrometers squared.

Immunohistochemistry
Labeling of relevant proteins was performed as follows. Cell cultures were grown in slide flasks (Nunc), fixed in 4% PFA for 15 min at room temperature, and transferred to blocking solution (1x PBS containing 3% horse serum and 0.5% saponin; Sigma) for 15 min. For analysis of unpermeabilized cells, the blocking solution contained 1x PBS containing 3% horse serum only. Primary and secondary antibody incubations were performed in blocking solution using standard techniques. Negative control samples without primary antibody were included and produced negligible background fluorescence.

Imaging
Images were captured on either a Zeiss Axioplan 2 fluorescence microscope equipped with an Axiocam HRm CCD camera or an LSM510 confocal head attached to an Axiovert microscope. In both cases, a Plan-Apochromat 63x-1.4 oil immersion objective with Zeiss Immersol was used. Fluorescence images were captured with Axiovision 3.1 or 4.2 and confocal imaging with Zeiss LSM510 imaging software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dysferlin localization in C2C12 myotubes
The subcellular distribution of dysferlin during the early stages of myogenesis has not been described so far. For this purpose, we used detection by immunofluorescence in differentiated C2C12 myotubes. Dysferlin expression in C2C12 begins at 2–3 days after induction of differentiation whereas myoblast fusion is detectable from the first day of differentiation onwards, indicating a role of dysferlin in the later stages of myotube formation and fusion (19 , 26) . Dysferlin was highly concentrated at the site of contact between two myotubes or a myoblast and a myotube (Fig. 1 A, arrows), where it colocalized with Bin1 (Fig. 1A ), a marker for the T-tubule system also implicated in myotube fusion (27) . Colocalization was also detected in regions in close proximity to but distinct from the plasma membrane, most likely representing beaded invaginations of the plasma membrane as part of the early T-tubule system (left-hand side of the cell depicted in Fig. 1A ). Unlike in mature skeletal muscle fibers, dysferlin failed to localize to the plasma membrane of myotubes, even after 6 days of differentiation (Fig. 1B ). Instead, from the third day of differentiation, endogenous dysferlin was predominantly localized in an extensive internal reticular network (Fig. 1B, C ). Colocalization with Bin1 identified this intracellular network as the T-tubule system (Fig. 1C ). Cholesterol depletion by methyl beta-cyclodextrin impairs T-tubule formation in C2C12 myotubes (27 , 28) , and exposure of C2C12 myotubes to methyl beta-cyclodextrin disrupted the pattern of dysferlin and Bin1 immunoreactivity (Fig. 1D ), confirming an association of dysferlin with the T-tubule system in C2C12 myotubes. Taken together, these results suggest a function of dysferlin in fusion and T-tubulogenesis in developing muscle and demonstrate that the sarcolemmal localization of dysferlin occurs in later stages of differentiation.

Targeting to the T-tubule system requires the full-length protein
We then investigated whether specific domains of dysferlin are responsible for its localization. The expression of truncated dysferlin proteins (Fig. 2 A) by transient transfection in C2C12 myoblasts demonstrated distinct expression patterns (Fig. 2B ). Only the full-length dysferlin protein (GFP-DFL) was targeted to the T-tubule system and colocalized with Bin1. Colocalization with a marker for the Golgi apparatus was found for DSC-TM and 3'GFP-2 and partially with DSC-Tth [data not shown, and L. M. Cree, PhD Thesis (2004), Newcastle University, UK]. DSC-Xho showed a diffuse cytoplasmic localization and DSC-Xba localized to the nucleus. Furthermore, none of these mutant constructs localized to the plasma membrane or to areas of myotube fusion.

However, direct comparison of the transfected and nontransfected populations in the same cultures demonstrated no abnormalities in cellular architecture. The presence of elongated myotubes, as opposed to that observed in myoblasts lacking dysferlin (14) , indicated normal fusion and differentiation and the absence of a dominant negative effect of the mutant constructs on endogenous dysferlin. These results suggest that the various domains of the protein are all required for correct localization during differentiation.

Membrane repair in C2C12 myotubes requires extracellular Ca2+
As we found that dysferlin did not localize to the plasma membrane of differentiating myotubes, we investigated if dysferlin could still be involved in the cell repair mechanism at this stage. To investigate the response of dysferlin to wounding and membrane resealing in C2C12 myotubes, we designed a wounding assay involving the dispersion of glass beads (29 , 30) on a monolayer of differentiated C2C12 myotubes. Glass bead wounding led to intracellular accumulation of membrane impermeable rhodamine dextran and indicated membrane damage (Fig. 3 A). Viability of cells 5 h after wounding was demonstrated by a BrdU assay detecting DNA synthesis (Fig. 3B ), and additionally, by 7-AAD staining to detect dead cells (Fig. 3C ). These findings demonstrated successful resealing of the plasma membrane. The assay was further refined by performing glass bead wounding in the presence and absence of extracellular Ca2+. The higher rate of cell death in the population wounded in the absence of Ca2+ (Fig. 3C ) indicated that efficient membrane repair after bead wounding requires extracellular Ca2+. To further explore the potential of this assay with regard to quantification of membrane damage, we applied it in the presence and absence of poloxamer 188, a membrane sealing agent known to reduce membrane susceptibility to damage in vitro and in vivo (31 , 32) . Figure 3D shows that poloxamer 188 reduces the amount of dead cells after wounding as indicated by staining with 7-AAD (P=0.0005). These results indicate that this assay allows quantification of survival after membrane wounding.


Figure 3
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  		Figure 3. Membrane wounding induced by glass beads and Ca2+ dependent repair in C2C12 myotubes. A) Rhodamine dextran (red) accumulates in myotubes after bead wounding. Untreated cells (control) do not incorporate the membrane impermeable dye. Nuclei are labeled with DAPI. Scale bar = 50 µm, wide field fluorescence images. B) Viability of cells was demonstrated 5 h after bead wounding by a BrdU assay detecting DNA synthesis (green). Because differentiated myotubes have low mitogenic activity, the overall rate of BrdU positive cells is similarly low in the control and wounded population. However, BrdU positive myotubes incorporating rhodamine dextran (red) were detected (inset). Scale bar = 50 µm, wide field fluorescence images. C) Quantification of cell death on slides after bead wounding applying 7-AAD as a marker of cell death and measuring area of fluorescence in µm2. The amount of dead cells is higher in population wounded in the absence of extracellular Ca2+. D) The assay is utilized to investigate the effect of a membrane sealing agent, poloxamer 188 (P188), on membrane susceptibility to damage in the presence of extracellular Ca2+. As indicated by area of fluorescence for 7-AAD, treatment with P188 (0.5 mM) reduces the number of dead cells and therefore membrane susceptibility to damage compared to the nontreated group. Data shown in C and D are an average of 20 random microscopic fields per slide with 3 slides per group and 3 experiments.

These results therefore demonstrate that glass bead wounding is a reliable assay to test the Ca2+-dependent membrane repair machinery in C2C12 myotubes and that membrane damage is quantifiable.

Full-length dysferlin translocates to the plasma membrane after bead wounding
Although dysferlin was not localized at the sarcolemma in unwounded cells, bead wounding of C2C12 myotubes not only led to a focal enrichment of dysferlin close to the site of injury but also to a general rearrangement of dysferlin localization toward the plasma membrane (Fig. 4 A–E).


Figure 4
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  		Figure 4. Dysferlin translocation in C2C12 myotubes after bead wounding detected by immunofluorescence. All images are confocal sections through the middle of the cells. A) Dysferlin (green) translocates toward plasma membrane in a C2C12 myotube that has experienced membrane ruptures. Arrowheads indicate 2 visible rupture sites. B) Rhodamine dextran (red) is demonstrated within damaged C2C12 myotube and leaking out at visible lesions. C) Focal, dysferlin positive structures (green), which appear to have accumulated around wounding sites and at the plasma membrane, can be noted (arrowheads; insets of A and B). Scale bars = 5 µm in A–C. D) Wounding site can be identified by the presence of extracellular rhodamine dextran (arrowhead). E) Corresponding region of plasma membrane shows enrichment of dysferlin (green) and subsarcolemmal accumulation of dysferlin. Scale bar = 5 µm in D and E. F–I) Dysferlin (green) can be detected in focal accumulations at a site of wounding on a nonpermeabilized C2C12 myotube. Rhodamine dextran is shown in red (H and I are insets of F and G). Scale bar = 5 µm.

Wounding was indicated by intracellular rhodamine dextran and leakage at the site of dysferlin enrichment. In nonpermeabilized cells, dysferlin labeling was only detected at the site of a membrane wound in focal accumulations (Fig. 4F-I ). No differences were detected when analyzing the cells immediately, 3, or 5 min after wounding. This effect was a specific response of dysferlin to the membrane injury, as it was not seen with other proteins involved in T-tubule development like caveolin-3 (Fig. 5 A), Bin1 (Fig. 5B ), or a Golgi-marker (data not shown). The translocation of dysferlin was Ca2+ dependent with no translocation occurring in the absence of extracellular Ca2+ despite the generation of dysferlin positive accumulations in the cytoplasm (Fig. 5C ). To investigate this finding further, we performed the wounding assay in C2C12 myotubes transfected with the full-length dysferlin construct and demonstrated a similar pattern of translocation to the plasma membrane as seen with endogenous dysferlin (Fig. 5D, E ). In contrast to the full-length dysferlin construct, none of the dysferlin mutant constructs translocated to the plasma membrane after wounding, which indicates that the translocation of dysferlin also requires the full-length protein (Fig. 6 ).


Figure 5
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  		Figure 5. Dysferlin translocation after wounding is specific and Ca2+ dependent. All images are confocal sections through middle of cells. A) GFP caveolin-3 does not translocate to the membrane after wounding in C2C12 myotubes. A wounding site is indicated by arrowhead, and wounding is indicated by intracellular rhodamine dextran. Scale bar = 10 µm. B) Bin1 does not translocate to membrane after bead induced membrane injury in C2C12 myotubes; wounding site is indicated by arrowhead. Scale bar = 10 µm. C) Endogenous dysferlin translocation requires extracellular Ca2+. Chelation of extracellular Ca2+ with EGTA inhibits translocation of dysferlin to membrane as a response to membrane damage despite focal accumulation in cytoplasm. Scale bar = 10 µm. D, E) GFP fluorescence of transiently transfected full-length dysferlin construct shows enrichment in plasma membrane (arrowheads in D) and appearance of dysferlin positive vesicles as a response to bead wounding E. Scale bar = 20 µm in D and 5 µm in E.


Figure 6
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  		Figure 6. None of the truncated dysferlin constructs translocates as a response to membrane wounding. GFP fluorescence of C2C12 myotubes expressing the constructs depicted in Fig. 2A in bead wounded cells as indicated by corresponding dextran fluorescence (red). Scale bar = 10 µm, wide field fluorescence images.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dysferlin is expressed at early stages of muscle development and localizes predominantly to the periphery of the myofibre in mature muscle (5) . Although dysferlin expression has been extensively analyzed in correlation with muscle disease, little is known about the trafficking of this protein at the earlier stages of muscle differentiation. In this study, we examined the subcellular localization of dysferlin in untreated and wounded C2C12 myotubes to study its role in early myogenesis. We found that although dysferlin is mainly expressed in the T-tubular network in developing muscle, it is able to translocate to the site of injury and plasma membrane as an immediate and specific response to membrane wounding. Our results demonstrate that 1) in the early stages of C2C12 myotube maturation dysferlin does not localize to the sarcolemma but is expressed at the T-tubule system and at sites of cell fusion, and 2) C2C12 myotubes are capable of resealing membrane ruptures in a Ca2+-dependent fashion involving translocation of dysferlin to the site of membrane injury. The full-length protein is required for both: correct localization and translocation to the membrane.

We have shown that in differentiated C2C12 myotubes dysferlin accumulates at the site of myoblasts fusing to myotubes. At this stage of muscle development, dysferlin is predominantly expressed in the T-tubule system of multinucleated myotubes, and the full-length protein is required for its correct localization. T-tubules form an intracellular membrane system that penetrates the myofibre allowing the action potential to reach the muscle fiber interior and to facilitate excitation contraction coupling (33) . T-tubules develop from beaded tubular invaginations of the plasma membrane (Fig. 1A ) and partly from longitudinal, cytoplasmic profiles, also termed beaded tubules, which are formed of strings of caveolae (34 35 36) . Caveolin-3 and Bin1 have been shown to be involved in T-tubulogenesis (27 , 37 , 38) . Bin1 is a conserved member of the BAR family of genes and has been implicated in myoblast differentiation and membrane deformation (27) . In mice lacking either caveolin-3 or Bin1, T-tubules are abnormal but not absent (39 , 40) , suggesting that additional factors are likely to contribute to T-tubule development (27) . Ampong et al. (7) suggested a role for dysferlin in fusion of caveolin-3 containing vesicles with T-tubules on the basis of an interaction of dysferlin with the dihydropyridine receptor in mature skeletal muscle and the known interaction of dysferlin with caveolin-3. This theory is further supported by the partial colocalization of dysferlin and caveolin-3 in early myogenesis (41 , and our unpublished observations), our findings that dysferlin colocalizes with Bin1 in C2C12 myotubes and the requirement for advanced mechanisms of membrane fusion and organization in the process of T-tubule biogenesis. Bin1 was highly abundant at fusion sites of myotubes in our study, colocalizing with full-length dysferlin. This supports a possible joint role of Bin1 and dysferlin in membrane fusion processes in developing muscle. As dysferlin and Bin1 expression both start at similar time points (day 2–3 in the differentiation process; refs. 19 , 26 , 27 ) and fusion already takes place before this, their role in fusion appears to be important in the later stages of differentiation. This is reflected by the fact that Bin1 and dysferlin deficient myotubes display impairment but not absence of fusion and differentiation (14 , 27) . Their T-tubule localization has important implications in view of the fact that the T-tubule system is involved in the generation of membrane for the elongation and repair of the sarcolemma and to "react" in regenerating fibers (42) . Furthermore, it serves as a membrane source for membrane bound vacuoles, and in skeletal muscle of dysferlinopathy patients, subsarcolemmal vacuoles contiguous with the T-tubule are one of the ultrastructural hallmarks (16) . This could be explained by an abnormal membrane fusion/budding mechanism in dysferlin deficient muscle, since in normal muscle the T-tubule system initiates the formation of and serves as a membrane source for autophagic vacuoles and vesicles (42) .

Various lines of evidence have suggested that dysferlin is involved in the global process of membrane repair (6 , 14 , 16) . This observation, however, has also exposed the fact that the mechanism and components of membrane repair in muscle remain relatively poorly understood, despite the fact that skeletal muscle has to have very highly developed systems to repair contraction induced fiber damage (43) . Membrane repair requires efficient membrane fusion. This is an active process whereby influx of extracellular Ca2+ activates a cascade of events ultimately leading to homotypic fusion of intracellular vesicles and consequently fusion of these endomembrane compartments with the plasma membrane to create a patch: the patch hypothesis (44 , 45) . This process is analogous to the synaptic vesicle fusion machinery depending on synaptotagmin (46) to which dysferlin has structural and biochemical similarity. This led to the hypothesis that dysferlin acts as a mediator in vesicle fusion (6 , 7 , 11 , 14 , 19) , which has so far remained unproven. Our results strengthen this hypothesis, demonstrating for the first time the focal accumulation of dysferlin positive structures in a Ca2+-dependent manner at and around the sites of injury as a consequence of membrane wounding. It is generally thought that focal enrichment of dysferlin may be vesicular in nature due to the following reasons: 1) the homology of dysferlin to fer-1 in C. elegans with an involvement in vesicle fusion (20 , 21) ; 2) the structural similarity of dysferlin to synaptotagmin; 3) its biochemical properties with six C2 domains render the protein highly lipophilic, and C2 domains are known to facilitate Ca2+-dependent binding of proteins to phosholipid membranes (23) ; 4) disruption of the T-tubule system leads to enrichment of dysferlin in the derived vesicles/endosomes (Fig. 1D ); and 5) crude biochemical fractionation of muscle or C2C12 myotubes consistently demonstrates dysferlin only in membrane fractions (7 and our unpublished data).

While in mature skeletal muscle the source of dysferlin accumulation at the wounding site is unknown, we have shown for the first time that in early myogenesis dysferlin accumulating at wounding sites is derived from a T-tubule localization. Therefore, the T-tubule system appears to serve as a pool of dysferlin positive endomembrane in the process of membrane repair. This response requires the full-length dysferlin protein, and this mechanism is in place in an early stage of development, as T-tubules have not yet orientated into the typical transverse pattern (47) . The first C2 domain of dysferlin (C2A) has been shown to bind phospholipids in a Ca2+-dependent manner (26) . In synaptotagmin harboring two C2 domains, one domain binds phospholipids, whereas the other C2 domain acts in protein-protein interactions. Our results suggest that to function in membrane fusion and repair, all six C2 domains are required, most likely mediating interactions with other proteins like annexin 1 and 2 and AHNAK (8 , 11) . In vitro assays have not been able to confirm this yet as the biochemical properties of dysferlin with its six C2 domains render the protein highly interactive and hamper its purification, but our results indicate the requirement for all the C2 domains to be present for correct dysferlin localization and translocation. This could have implications for therapeutic strategies for dysferlinopathies where the goal is not to restore the full-length protein such as the generation of minidysferlins or exon skipping.

The cell wounding system we have established is a robust, inexpensive, and reproducible assay to analyze the effects of membrane wounding in a high number of cells and will be a valuable tool for the exploration of possible therapeutic agents aiming to promote membrane stability in MD in vitro, as we have shown for the triblock copolymer poloxamer 188 (Fig. 3D ). Glass bead wounding causes repeated contact of the beads with the cell membrane and could therefore serve as a model for the shearing forces that are generated during repeated cycles of contraction and relaxation in skeletal muscle. It resulted in membrane rupture and led to translocation of dysferlin to the membrane. Dysferlin translocation was specific, dependent on the presence of extracellular Ca2+ and the full-length protein, and dysferlin was detectable in focal accumulations and in stretches of plasma membrane highly indicative of a function in vesicle fusion as proposed in the patch hypothesis (44) .

Taken together, our results provide further evidence for a specific role of dysferlin in membrane fusion and repair and that dysferlin is involved in this mechanism in early stages of development. It is possible that mechanical stressors resulting in membrane wounding are responsible for the sarcolemmal localization of dysferlin during the course of maturation. Identification of factors modifying the role of dysferlin in membrane repair will contribute to the further understanding of the basic biological mechanisms of membrane fusion and repair in muscle, and they will be potential candidate genes for other muscle diseases with yet unidentified etiology.


   ACKNOWLEDGMENTS
 
We thank Lisa Hodgson (Institute of Human Genetics, University of Newcastle upon Tyne, UK) for support regarding imaging techniques and Dr. Mark Hornsey (Institute of Human Genetics, University of Newcastle upon Tyne, UK) for critical reading of the manuscript. The Ph.D. thesis by Dr. Lynsey Cree provided useful background information. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KL 1868/1–1 to L. Klinge, and by grants provided by the Muscular Dystrophy Campaign, Association Francaise contre les Myopathies, and the Jain Foundation).

Received for publication November 9, 2006. Accepted for publication January 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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